Method of producing IGF-1

ABSTRACT

Ala-Glu-IGF-I is a novel compound which exerts IGF-I activity and is a precursor for the preparation of IGF-I. Ala-Glu-IGF-I may by converted to IGF-I by renaturation after recombinant production in E. coli under specified conditions and then cleaving Ala-Glu from the IGF-I.

This application is a continuation application Ser. No. 07/746,827,filed Aug. 19, 1991, now abandoned.

The present invention relates to novel human insulin-like growth factor(IGF-1) having an amino terminal extension, a DNA sequence coding forIGF-1 having such an amino terminal extension, a recombinant plasmidcomprising DNA coding for human IGF-1 having an amino terminalextension, a transformant microorganism containing such recombinantplasmid, a method for the renaturation of AlaGlu-IGF-1, a method forproducing human IGF-1, and use of an amino terminal extended IGF-1 forthe preparation of mature IGF-1. The invention is particularly suitablefor use in large scale production of IGF-1.

BACKGROUND OF THE INVENTION

The insulin-like growth factors (IGFs) constitute a family of proteinshaving insulin-like and growth stimulating properties. The peptides havebeen purified from plasma, and their structure and chemical propertieshave been determined. The IGFs show close structural homology withproinsulin and elicit similar biological effects (Rinderknecht,E. andHumbel, R. E.: J. Biol. Chem., 253, 2769-2773 (1978), Zapf,J. et al.:Curr. Topics in Cell Reg., 19,257-309 (1981), Humbel,R. E.: HormonalProteins and Peptides, 12, AP 56-69 (1984), van Wyk,J. J.: HormonalProteins and Peptides, 12, AP 81-125 (1984)). Although being members ofthe same family of proteins, the two main forms of IGFs are ratherdifferent with respect to their chemical characteristics. IGF-1 is abasic peptide (pI 8.4) and shows a 43% homology with proinsulin(Rinderknecht,E. and Humbel,R. E.: J. Biol. Chem., 253, 2769-76 (1978).IGF-2 is an almost neutral peptide (pI 6.4) shows a 60% homology withIGF-1.

IGF-1 consists of 70 amino acids, the sequence of which has beendetermined by Rinderknecht,E. and Humbel,R. E.: J. Biol. Chem, 253,2769-73 (1978). The physical-chemical parameters for IGF-1 have beenstudied extensively by several groups. The material used for thesestudies was primarily isolated from human plasma by a variety ofconventional chromatographic methods. These include initial plasmafractionation and further precipitation of other proteins using acidethanol extraction to remove binding proteins and other plasma proteins.The acidified IGF-1-enriched fraction was then subjected to cationexchange chromatography, and several steps of reversed phase andisoelectric focusing resulting in the collection of a few μg of purifiedIGF-1.

IGF-1 and to a lesser degree IGF-2 are found in plasma, but only a minorfraction is present in a free form. Specific binding proteins of highmolecular weight having very high binding capacity for both IGFs act ascarrier proteins or as modulators of IGF functions (Holly,J. M. P. andWass,J. A. H.: J. Endocrinol, 122,611-618 (1989), Ooi,G. T., andHerrington,A. C.: J. Endocrinol., 118, 7-18 (1988)). The IGFs exert aninsulin-like effect when present in high amounts although only to anextent of 1% of that of insulin. The somatic function of IGF-1 isformulated in the so-called "Somatomedin Hypothesis", suggesting thatgrowth hormone released from the pituitary gland exerts its effect bystimulating IGF-1 release from the liver, which IGF-1 then mediates thesomatogenic actions via binding to cell surface receptors in the targettissues (Salmon,W. D. Jr., and Daughaday,W. H.: J. Lab. Clin. Med., 49,825-836, (1957). The principal local biological function of IGFs is as apotent mitogenic agent, although the effect is weaker than that measurede.g. for PDGF and FGF (Froesch et al.: Ann. Rev. Phyiol. 47, 443-467(1985). Together with PDGF, IGF-1 shows synergistic biological effects(Kato et al.: Eur. J. Biochem., 129, 685-690 (1983), Stiles et al.:P.N.A.S., USA 76, 1279-1283 (1979)). The principal biological effects ofIGF-1 are thus considered the function of propagating mitogenicresponses, and of maintaining growth initiated by other factors underproperly controlled conditions (O Keefe et al.: Mol. Cell. Endocrinol.,31, 167-186 (1983) .

The potency of IGF-1 as a mitogenic factor and the vast potential usethereof in e.g. wound healing and in nitrogen metabolism has encourageda great number of biopharmaceutical companies to try to express IGF-1 invarious organisms due to the small amount present in human plasma.Production of IGF-1 in yeast is favored by a very easy purification of acorrectly folded polypeptide. However, there are certain significantdrawbacks associated with the use of these expression systems. The yieldof fermentation is low (few mg per liter of fermentation broth), and therisk of obtaining O-linked glycosylated forms of the molecule issignificant (Gillerfors et al.: J. Biol. Chem., 264, 2748-53), (1989).

Expression in bacteria has until now been the most successful approachgiving high yields of IGF-1 and IGF-2, but mostly in cell associatedforms. It has also been tried to express IGF-1 fused to a stabilizingpeptide. Such a peptide is normally of the same size as IGF-1 itself,and may have physico-chemical properties facilitating the purificationof the resulting protein. The fusion protein is cleaved by eitherchemical or enzymatic cleavage, leaving the mature protein forisolation.

Extraction of the recombinant peptide (fusion peptide) is normallyassociated with a denaturating step followed by in vitro renaturation ofthe extracted IGF-1, resulting in the native conformation of IGF-1.

In the bacterial systems described so far, yields of from 1 to 1.5 gramsper liter of fermentation broth have been described for biosyntheticexpression of hGH. However, despite the high yield of expression onlyminor amounts of native IGF-1 is obtained because of uncontrolledformation of intra and inter molecular disulphide bridges. Therefore,bacterial expression of products containing such disulphide bridges hasto be followed by in vitro renaturation during the later purification ofthe polypeptides which normally results in extensive losses of product.

The object of the present invention is primarily to overcome theproblems described in the prior art with respect to producing IGF-1 inmicroorganisms in high yields.

As used in the present context, the expression "folding" is used todesignate the process of establishing the tertiary structure of theprotein, including the formation of the intramolecular disulphide bonds.

The expression "Renaturation" is used to designate the establishing ofthe native tertiary structure of the protein.

The expression "Denaturing" is used to designate the break-down of thetertiary structure of the molecules disrupting the disulphide bondsand/or uncoiling of the protein.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to human IGF-1 having an amino terminalextension AlaGlu.

The amino terminal extension allows high expression of the extendedIGF-1, easy renaturing or folding of the IGF-1 and easy and completecleavage thereof using the enzyme Dipeptidyl Amino Peptidase (DAP-1, EC3.4.14.1) to form the mature IGF-1.

Thus, it has surprisingly been found that the amino terminal extensionAlaGlu overcomes the problems of the prior art. Furthermore, it ispossible to purify the IGF-1 having correct N-terminus from the reactionmixture.

The AlaGlu-IGF-1 may be isolated as an intermediate in a purity >80%,preferably >90% and shows, when correctly folded, biological activity ofIGF-1.

The present invention also relates to a DNA sequence coding for IGF-1having the amino terminal extension AlaGlu. This short extension hasbeen applied in the expression of IL-1β and hGH but has not beenproposed for the expression of IGF-1.

The part of the DNA encoding IGF-1 may be cDNA, chromosomal DNA orentirely or partly synthetic DNA wherein any remainder of the DNA may bethe corresponding cDNA or chromosomal DNA.

The nucleotide sequences used for the amino terminal extension and anysynthetic part of the DNA encoding IGF-1 are preferably chosen to givethe preferred codons for the chosen microorganism.

The expression of the amino terminal extended AlaGlu-IGF-1 results in anexpression level of 1.5 g/liter (0.2 mM) AlaGlu-IGF-1 prior toextraction. This is approximately 4 to 5 times the specific expressionobtained in systems applying longer fusion proteins. However, not allshort-chain aminoterminal extentions give rise to high levels ofexpression. Expression of e.g. MetGluAlaGlu-IGF-1, (AlaGlu)₆ -IGF-1, orIGF-1 itself all give rise to levels of expression 10 to 25 times belowthe level obtained expressing AlaGlu-IGF-1.

The DNA encoding AlaGlu-IGF-1 may be prepared from a cloned DNA sequenceencoding the 70 amino acids of IGF-1 which is coupled with the followingsynthetically produced, dual-stranded DNA sequence in such a manner thatthe 3' end of the +strand is coupled to the +5' end of the gene encodingIGF-1 and the 5' end of the synthetic DNA strand is coupled to the 3'end of the gene by blunt end ligature

    +5' CGATG GCT GAA

    -3' TAC CGA CTT

wherein the two first nucleotides of the +strand are a ClaI restrictionsite overhang and the following nucleotide sequence code for the aminoacids MetAlaGlu.

According to a further aspect of the invention a gene being apt forindustrial production, coding for an amino terminally extended IGF-1ensuring the formation of a stable product, and furthermore to thesecretion thereof into the periplasmic space or directly into the mediumis provided.

According to a third aspect, the invention relates to a recombinantvector comprising DNA-coding for human IGF-1 having the amino terminalextension AlaGlu. The vector may be in the form of a plasmid comprisinga promoter upstream the DNA coding for AlaGlu-IGF-1 and preferablycomprises the temperature sensitive promoter λAPR coupled directly tothe DNA coding for AlaGlu-IGF-1 and the OmpA signal sequence providingfor the secretion of the AlaGlu-IGF-1 into the periplasmatic space ordirectly into the medium. This vector has been demonstrated to be aptfor industrial production when inserted in E.Coli.

According to a fourth aspect the invention provides a transformedmicroorganism comprising said recombinant plasmid.

According to a fifth aspect, the invention relates to a method for therenaturation of AlaGlu-IGF-1 wherein AlaGlu-IGF-1, in a substantiallydenatured and reduced form, is folded in the presence of a mercaptoreagent in its reduced form in a buffered aqueous solution. Therenaturation takes place during the controlled change of the redoxpotential from ÷40 mV to +20 to +40 mV by dialysis against a buffercontaining from 20-40% v/v ethanol over a period of up to 5 hours at aprotein concentration of 0.1 to 0.6 mg/ml and a conductivity of 0.15 to0.3 mS at ambient temperature and a pH of from 7.5 to 10.0 and finallyacidifying the mixture to a pH below 5.

The mercapto reagent used in the method of the invention may be anymercapto reagent which does not form unwanted by-products, e,g.2-mercaptoethanol, cysteine, cysteamin, preferably cysteine.

In accordance with the invention, the mercapto reagent is used in aconcentration of from 0.01-10 mM, preferably a concentration of 0.1-5 mMand most preferred a concentration of 1 mM.

The buffered aqueous solution in which the folding or renaturation ofthe the invention is carried out is buffered to a pH in the intervalfrom 8.0-10.0, preferably a pH of 9.0.

In some cases it is preferred to carry out the renaturation according tothe invention in the presence of ethanol in an amount of up to 40%,preferably about 25%. Such addition increases the yield of the method,and the optional beneficial effect of ethanol is easily demonstrated bysimple preliminary experiments.

According to a sixth aspect the invention relates to a process forproducing IGF-1 comprising

i) expression of an amino terminal extended IGF-1 in a microorganismtransformed with an expression vector comprising an inducible promotercoupled directly to the DNA coding for human IGF-1 having an aminoterminal extension and a signal sequence, providing for secretion of theexpressed product,

ii) extraction of amino terminal extended IGF-1 at pH>6.0 using highconcentration of urea in the presence of a reducing agent and optionallya chelating agent,

iii) subjection of the extract to chromatography on an anion exchangegel selected from the group consisting of DEAE, DE and FF-Q using thesame buffer as in step ii) adjusted to 1.8-2.2 mS and pH 7.8-8.2,

iv) renaturation of the isolated amino terminal extended IGF-1 changingthe redox potential of the reaction mixture linearily from -40 mV to +20to +40 mV by dialysis against a buffer containing from 20-40% v/vethanol over a period of up to 5 hours keeping a protein concentrationfrom 0.1 to 0.6 mg/ml and a conductivity of 0.15-0.3 mS, at ambienttemperature and a pH of from 7.5 to 10.0 and finally acidifying themixture to a pH below 5,

v) cleaving the amino terminal extension using DAP-1,

vi) isolation of the renatured authentic human IGF-1 from aminoterminally extended IGF-1 using RP-HPLC and cation exchangechromatography, and

vii) gel filtrating and lyophilizing the isolated human IGF-1.

The method of the invention may be carried out by expressing the aminoterminally extended IGF-1 in a microorganism such as a grampositivebacterium or a gramnegative bacterium such as Esherichia, preferablyE.Coli which is a very convenient host due to its industrialapplicability.

According to the invention the promoter may be a temperature sensitivepromoters such as λ_(PR), λ_(PL) or λ_(PR), and the signal sequence maybe LamB, OmpA or OmpF sequences in order to ensure a proper expressionand secretion.

The AlaGlu-IGF-1 is expressed in a high level using the λ_(PR) promoterdirectly coupled to the gene coding for AlaGlu-IGF-1, and the OmpAsignal sequence provides for secretion of the protein in a stable form.

The extraction of AlaGlu-IGF-1 is carried out under reducing anddenaturing conditions using urea, reducing agents such as cysteine,2-mercapto-ethanol or dithiothreithol and optionally chelating agent(s)such as EDTA in order to destroy the tertiary structure imposed on theAlaGlu-IGF-1 by the express ion system.

The extraction of the AlaGlu-IGF-1 in a totally denatured form ispreferably carried out using a buffer containing 7M urea, 50 mM Cys atthe isoelectric point (8.4) or pH 8.0.

The majority of contaminating proteins originating from E.coli areremoved from the reduced and denatured AlaGlu-IGF-1 by anion exchangechromatography at the isoelectric point. This chromatography ispreferably carried out at a conductivity of 2.0 mS at pH 8.0 and ispreferably carried out as batch chromatography on DE-52 (Whatman)repeated on Fast Flow Q sepharose® (FF-Q®).

The reduced and denatured AlaGlu-IGF-1 is renatured into stable,AlaGlu-IGF-1 being correctly folded.

The renaturation process is carried out using dialysis under control ofthe redox potential, pH, the protein concentration and the temperaturein the presence of chelating, denaturing and mercapto reagents, and inthe presence of organic solvents.

The dialysis buffer is characterized by the presence of small amounts ofa reducing agent (e.g. DDT, Cys, or 2-mercapto ethanol), a chelatingagent, an organic compound to interact with hydrophobic bonding (such astert-butanol, 2-propanol, ethanol, methanol), and finally a buffersubstance to maintain a well defined pH interval, typically 8.8 to 9.0.In one embodiment, the following buffer composition was used: 50mMtris-HCl, 1 mM Cys, 2 mM EDTA and 25% ethanol at pH 9.5.

The renaturation is preferably carried out by changing the redoxpotential linearily from about -30 mV to between +25 and 30 mV over aperiod of 3 hours. The redox electrodes are calibrated using ahydroquinone solution having a well defined redox potential of 463 mV.

It is preferred to carry out the renaturation at a conductivity of 0.2mS concentration of proteins of 0.1 to 0.4 mg/ml and a pH from 8.5 til9.5 especially preferred a pH of 8.5 to 9.0 as measured in the bufferbefore addition of ethanol in a concentration of from 20 to 40% (v/v)and at a temperature of from 20° til 22° C.

According to a most preferred aspect, the renaturation of AlaGlu-IGF-1is carried out passing the polypeptide through a hollow fiber device(Nephross Presto,H.F., Organon Teknika) at a flow rate from 25 to 250ml/min, preferably about 100 ml/min while changing the redox potential,until the process is terminated by adding 5M acetic acid to a pH below5, preferably pH 4.0.

The resulting solution is then concentrated by cation exchangechromatography in the presence of organic solvents before cleaving offthe amino terminal extension. The cation exchange is preferably carriedout using a strong ion exchange material such as FF-S® or FF-SP® in thepresence of 10-40% alcohol such as methanol, ethanol, a propanol or abutanol, preferably 25% ethanol and using a pH gradient between 3.5 and7.5.

The cleavage of renatured AlaGlu-IGF-1 is preferably carried out usingthe exopeptidase (DAP-1) giving a quick and efficient cleavage of theIGF-1 precursor. The exopeptidase is preferably used in a concentrationof 0.08 units DAP-1 per mg protein in the presence of NaCl adjusting theprotein concentration to approximately 1 mg/ml in a 40 mM acetate bufferat pH 4.0 and 37° C.

Reversed phase HPLC purification of IGF-1 after cleavage using DAP-1 maybe performed on a RP18-column either of a commercial type (Lichrosorb)or custom made C18 silanoyl RP18 column. Preferably a 15 μm Novo NordiskA/S silanoyl-RP18 column eluted at pH 3.0 in a 0.1M Na-phosphate bufferusing a 30-50% ethanol linear gradient is used.

Any IGF-1 not being correctly folded will be removed by reversed phasechromatography on a RP18-column either of a commercial type, e.g.Lichrosorb, or custom made C₁₈ silanoyl RP18 column using ethanol os themobile phase and remaining AlaGlu-IGF-1 will be removed using cationexhange chromatography starting at acid conditions and applying apH-gradient from pH 4.0 to pH 7.0 using the same buffer system as in theabove cation exchange procedure.

The purified IGF-1 is then subjected to lyophilization andgelfiltration. The lyophilized IGF-1 is taken up in 7 M urea, andapplied to a G50F GPC Sephadex® column equilibrated in 0.1M acetic acidat 4° C. The purified material is stored at 4° C.

Finally, the highly purified IGF-1 is subjected to dialysis andgelfiltration and lyophilization to form a lyophilized, chemicallystable powder.

Yet another aspect of the invention is a partial purification of theamino terminally extended IGF-1 in a fully denatured and reduced form.

A selective separation of amino terminally extended IGF-1 from IGF-1having the correct amino terminus and amino acid sequence is obtainedusing cation chromatography, after cleaving the aminoterminal extensionusing DAP-1 in accordance with the principle described by Dalboege, H.et al. FEBS Lett. 246 (1,2) 89-93, 1989.

The removal of incorrectly renatured (folded) forms of IGF-1 by reversephase HPLC is obtained using an octadecyl-dimethyl-silyl-substitutedsilica matrix comprising 15μ spherical particles having 300 Å porediameters.

The characterization of the renatured IGF-1 with respect to the correctpositioning of the disulphide bridges was carried out using twoenzymatic cleavages followed by peptide mapping and sequence analyses.

The process of the invention has the further advantage that it can beupscaled directly.

A valuable aspect of the invention is the extraction procedure givingvery high yield (close to 100%) of AlaGlu-IGF-1. The amino terminallyextended IGF-1 is then purified using a specific FF-Q® anion exchangechromatography step at pI conditions under which AlaGlu-IGF-1 is notbound to the resin, whereas proteins originating from E.Coli and apolypeptide exibiting physical-chemical characteristics similar to thatof AlaGlu-IGF-1, are removed under these specific conditions. Anothervaluable aspect of the invention is the specific renaturation step. Thisstep is characterized by the continuous control of redox potential, ionstrength and temperature during the renaturation of AlaGlu-IGF-1.

It has been found that modifications of the presequence drasticallychanges the conditions for renaturation.

The invention further provides a new concept for the cleavage of anN-terminal extension of IGF-1 using DAP-I (EC 3.4.14.1.), andapplication of a mild denaturation of the correctly folded IGF-1 priorto the final gel permeation chromatography step.

Thus, the invention apply a series of unique conditions which takentogether improves the quality of the product and yield of thedown-stream process following the fermentation.

The process may be scaled up to amounts in the order of grams per run,and a high degree of purification, illustrated by the low content ofproteins originating from E.Coli (<1 ppm), is obtained.

The product is shown to have an amino acid composition, amino acidsequence, disulphide bridging and peptide mapping identical to thoseseen for IGF-1 purified from human plasma. The product is furthercharacterized by showing one single band after SDS-polyacrylamide gelelectrophoresis, and on native gels. Finally the product ischaracterized to be more than 95% pure by analytical reversed phase HPLCand GPC.

Furthermore, the invention relates to the use of IGF-1 having the aminoterminal extension AlaGlu for the preparation of mature IGF-1.

The biological activity of the product of the method of the invention isequal to that of native IGF-1 purified from plasma. The product may e.g.be used for the treatment of wounds during wound healing, healing offractured bones, metabolic disorders, and Diabetes type II (NIDDM). TheIGF-1 may be formulated in any manner known per se for the formulationof pharmaceutical preparations or administration forms comprising IGF-1.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is described more in detail below with reference to thedrawings in which

FIG. 1 shows the cloning strategy of a vector coding for Ala-Glu-IGF-1,

FIG. 2 shows a HPLC diagram of partly purified Ala-Glu-IGF-1 in itsdenatured and reduced form,

FIG. 3 shows a HPLC diagram of partly purified Ala-Glu-IGF-1 in itsrenatured form,

FIG. 4 shows the analytical HPLC diagram of >95% pure IGF-1,

FIG. 5 shows the DNA sequence of pHD 147 encoding IGF-1.

FIG. 6 shows a RP-HPLC diagram of AlaGlu-IGF-1 after renaturation byhollow fiber dialysis,

FIG. 7 shows a RP-HPLC diagram of AlaGlu-IGF-1 after renaturation bydilution,

FIG. 8 shows a RP-HPLC diagram of AlaGlu-IGF-1 after renaturation bydesalting, and

FIG. 9 shows a RP-HPLC diagram of AlaGlu-IGF-1 after renaturation bydialysis.

The invention is further explained with reference to the specificExamples.

EXPERIMENTAL PART EXAMPLES Example 1

Construction of IGF-1 gene.

A synthetic gene for MetGlu-IGF-1 was constructed on basis of thepublished amino acid sequence (Jansen et al. 1983). Using optimum E.Colicodons, the gene was constructed from synthetic fragments which,following annealing and purification on gel, was cloned stepwise betweenunique restriction sites in appropriate cloning vectors (FIG. 1).

Construction of plasmid pHD145 (plasmid encoding aa 1-30 ofMetGlu-IGF-1):

Preparation of linker: 0.4 μg of each of the linkers upper a, lower aand upper b were kinated separately by adding 1 μl 10×kinase buffer, 1μl 10 mM ATP, 1 μl T4 PNK (10 units), 6 μl H₂ O. Incubation at 37° C.for 30 min. The mixture was then heated to 65° C. for 10 min.

10 μl kinased upper a and 10 μl kinated lower a were annealed by heatingto 100° C. for 3 rain and cooling on ice.

10 μl kinased upper b was annealed to 0.4 μg lower b (not kinated) byheating to 100° C. for 3 min. and cooling on ice.

Ligation of linker: 20 μl a, 11 μl b, 4 μl 10×ligase mix, 1 μl ligase(0.5 unit), 4 μl H₂ O were mixed and incubated at 15° C. for 2 hours.The mixture was run on a 1.8 % low melting agarose gel. A fragment of100 bp was isolated and purified using elutip. After precipitation, thefragment was dissolved in 50 μl H₂ O. 2 μl were taken out and checked on2% agarose gel.

Preparation of plasmid:

The above purified linker was then cloned in pBR322 digested withClaI/BalI. In order to facilitate the digestion of pBR322 with Bali itis necessary to culture this plasmid in a not methylating strain ofE.coli. Hence, pBR322 was transformed into the bacteria strain E.coliGCl17, which is DCM minus and DAM minus. 10 μl pBR322 (from GCl17) wasdigested using 5 U ClaI/3 U Bali in a low salt buffer in a final volumeof 100 μl. The incubation was at 37° C. for 2 days and nights.

A DNA fragnent of about 1900 bp (plasmid fragment) was purified on 0.7%low melting agarose gel. After elution using elutip, the DNA wasdissolved in 40 μl H₂ O.1 μl aliquot was checked on gel.

Ligation of pHD145:

The following ligations were set up:

    ______________________________________                                                 A           B           C                                            ______________________________________                                        100bp DNA                                                                              3 μl     3 μl     3 μl                                      linker a + b                                                                  pBR322   1 μl     0.05 μl  0.05 μl                                   ClaI/BalI                                                                     T4 DNA   0.05 unit (1 μl)                                                                       0.05 unit (1 μl)                                                                       1 unit (1 μl)                             ligase                                                                        10 × lig mix                                                                     1 μl     1 μl     1 μl                                      H.sub.2 O                                                                              4 μl     4 μl     4 μl                                      ______________________________________                                                 Control A   Control B   Control C                                    ______________________________________                                        100 bp DNA                                                                             0           0           0                                            linker                                                                        pBR32    1 μl     0.05 μl  0.05 μl                                   ClaI/BalI                                                                     T4 DNA   0.05 unit   0.05 unit   1 unit                                       ligase                                                                        10 × lig mix                                                                     1 μl     1 μl     1 μl                                      H.sub.2 O                                                                              7 μl     7 μl     7 μl                                      ______________________________________                                    

All the ligations were incubated at 15° C. for 3 hours, transformed intoMC1061, plated on LB+ampicillin (LBA) agar plates on nitrocellulosefilter and incubated at 37° C. over night. The reading of the plates onthe next day showed that there was a greater number of colonies on thecontrol plates than on the plates to which was added 100 bp fragment.Hence, it was decided to screen for any positive transformants by DNAhybridization.

Colony hybridization:

A copy of each filter was replicated onto a fresh nitrocellulose filter,both filters were transferred to LBA plates and incubated at 37° C. overnight. The bacteria on one of the filters were lysated for 2 min. in0.5M NaOH, 1.5M NaCl followed by washing in 0.5M Tris, pH 8, 1.5 NaClfor 2 min., and 2×SSC for 2 min. The filters were dried, baked for 1hour at 80° C., and hybridized to a probe.

Preparation of probe:

1 μl (0.4 μg) IGF-1 upper a, 1 μl (0.4 μg) IGF-1 upper b, 100 μCi γ ATP(10 μl ), 2 μl 10×kinase buffer, 1 μl (5 unit) PNK, H₂ O ad 20 μl weremixed and incubated at 37° C. for 30 min. The baked filters were, afterprehybridization at 65° C., added to the above probe in a 50 mlhybridization volume. The hybridization was carried out at 60° C. overnight. The filters were washed in 2×SSC followed by wash in 0.1×SSC, 50°C., dried and placed on a film.

15 positive colonies were cultured in LBA at 37° C. over night. Analysisof miniprep. DNA using the enzymes ClaI/PvuII showed, after run on 1.5 %agarose gel, that all clones except clone 11 comprised the expected DNAfragment of 745 bp. In order to further analyze some of the clones ,i.a. for presence of the BalI/ClaI fragment of about 100 bp, the clones1, 2, 3, and 4 were transformed into E.coli GC117 and plated on LBA agarplates. A single colony was chosen from each of the four plates and usedfor propagating maxiprep DNA. Digestion of the four plasmid preparationsshowed that the constructions 1, 2, and 3 had the expected restrictionsites, but that clone 4 lacked the BalI site.

Construction of pHD146 (plasmid having sequence coding for aa 1-56 ofMetGlu-IGF-1):

100 μl maxiprep DNA from pHD145-1, 2, and -3, respectively, was digestedwith 20 unit Bali over night at 37° C. in low salt. After checkingdigestion on agarose gel, the DNA was digested with 20 unit PvuI overnight at 37° C. in high salt. After the digestion, the DNA wasfractionated on 0.7% prep. agarose gel. A DNA fragment of 750 bp wasisolated from each clone, elutipped, precipitated and dissolved in 20 μlH₂ O. 0.4 μg linker C+D upper+lower were annealed in 20 μl H₂ O atboiling for 5 min. and cooled on ice. The annealed linker was thenpurified on 2.5% prep. gel, a band of 70 bp was isolated, eluted fromthe gel using elutip, precipitated and dissolved in 20 μl H₂ O.

The purified linker was kinated by adding 1 μl 10×kinase buffer, 1 μl 10mM ATP, 5 unit PNK, and H₂ O to 10 μl followed by incubation at 37° C.for 1 hour.

20 μl pBR322 DNA were digested with 50 unit PvuI/50 unit NruI in highsalt in a final volume of 100 μl. After digestion, the DNA wasfractionated on a gel, and a fragment of about 2800 bp was isolated,purified on elutip, precipitated and dissolved in 20 μl H₂ O.

The below ligations A, B, control C, and control D were set up withfragments isolated from pHD145-1, -2, and -3, respectively.

    ______________________________________                                                                  Con-                                                          A     B         trol C  Control D                                   ______________________________________                                        10 × lig mix                                                                        2     μl 2    μl                                                                              2   μl                                                                             2     μl                         2800 bp fragment                                                                          0.5   μl 0.5  μl                                                                              0.5 μl                                                                             0,5   μl                         750 bp fragment                                                                           0.5   μl 0.5  μl                                                                              0.5 μl                                                                             0                                   70 bp kinated                                                                 linker c + d                                                                              10    μl 1    μl                                                                              0       0                                   H.sub.2 O   6     μl 15   μl                                                                              16  μl                                                                             16.5  μl                         T4 DNA ligase                                                                             0.1   U     0.1  U    0.1 U   0.1   U                             0.1 U/μl                                                                   ______________________________________                                    

The above ligations were incubated at 15° C. over night, transformedinto MC1061 and plated out on LBA plates on nitrocellulose filter. Fiveplates were used per ligation. After incubation over night at 37° C.,all the plates were replicated and treated as described above underconstruction of pHD145. 1 μl (0.4 μg) upper c and 1 μl (0.4 μg) upper dlabelled as described for pHD145 was used as probe. Hybridization andwashing conditions were as described above. After development of film,24 positive colonies were chosen and propagated to miniprep DNA in LBAmedium. The DNA was analyzed by digestion with NruI/EcoRI and run on1.5% agarose gel. The clones 3, 4, 5, 8, 13, 16, 18, 19, 20, and 21looked right. Clone 3 and 13 were selected for closer analysis andpropagated for maxiprep DNA.

The 24 isolated clones were isolated from the following ligations:

    ______________________________________                                        Ligation reaction  750 bp pHD145-                                                                            Plate no.                                      ______________________________________                                        1       A              1           b                                          2,3,4,5,6                                                                             B              1           a                                          7,8     B              1           c                                          9       A              2           b                                          10      A              2           c                                          11,12,13,14                                                                           B              2           c                                          15,16   B              2           e                                          17      A              3           c                                          18      A              3           d                                          19,20,21                                                                              B              3           c                                          22,23,24                                                                              B              3           d                                          ______________________________________                                    

Cloning of pHD147 (plasmid having the sequence encoding aa 1-70 ofMetGlu-IGF-1 (FIG. 1)):

50 μl maxiprep DNA from pHD146-3 and pHD146-13, respectively, wasdigested with 20 unit NruI and 15 unit PvuI in 55 μl final volume(medium salt). 10 μl pBR322 plasmid DNA was digested with 15 unit PvuIand 36 unit HindIII in 55 μl final volume (medium salt). Both digestionswere fractionated on 0.8% low melting agarose gel. From the clonespHD146-3 and -13 a 800 bp fragment was isolated, elutipped, precipitatedand dissolved in 50 μl H₂ O. From pBR322 a 3500 bp fragment wasisolated, elutipped, precipitated and dissolved in 50 μl H₂ O.

Linker IGF-1 e was annealed and purified on 2.5% prep. agarose gel asdescribed for the previous linkers.

A 50 bp fragment was isolated and dissolved in 50 μl H₂ O.

All the above fragments were checked on agarose gel.

Ligation of pHD147:

    ______________________________________                                                         A      B                                                     ______________________________________                                        pBR322 3500 bp fragment                                                                          1.5   μl  1.5   μl                                   pHD146-3 800 bp fragment                                                                         10    μl  10    μl                                   50 bp linker e     2     μl  0                                             10 × lig mix 1.5   μl  1.5   μl                                   T4 DNA ligase 1 U/μl                                                                          1     unit   1     unit                                    ______________________________________                                    

The ligation was carried out using the 800 bp fragment from pHD146-3 aswell as from pHD146-13. The ligations were incubated for 3 hours at 15°C., transformed into MC1061, and plated on LBA plates. The filters werereplicated, washed and hybridized as described above. 2 μl IGF-1 e lowerDNA was used as probe. 6 positive clones were selected from filterswhere DNA from clones pHD146-3 and pHD146-13, respectively was used.These clones were called pHD-1->6 (pHD146-3+linker e) and pHD147-7->12(pHD146-13 +linker e). Analysis of miniprep. DNA from the 12 clonesusing the restriction enzymes EcoRI/HindIII showed that all clones hadthe expected fragment. The clones 1 and 2, 7 and 8 were chosen for acloser analysis with digestion with the restriction enzymesHindIII/ClaI, PvuII/BamHI, PstI/BamHI. It appeared that all the cloneshad the expected restriction pattern. The clones pHD148-1 and pHD147-7were propagated to maxiprep. DNA. Simultaneously miniprep. DNA wasisolated from the clones 1, 2, 7, and 8 digested with the restrictionenzymes EcoRI, HindIII, and a 220 bp fragment was isolated, cloned inM13, MP18 and MP19 and subjected to sequence analysis. The DNA sequenceanalysis showed that the clones comprised the expected DNA sequence.

Example 2

Cytoplasmic production of MetGlu-IGF-1

The plasmid pHD148-1 was digested with HindIII in the presense of KlenowDNA polymerase and dNTP, phenol extracted, precipitated and digestedwith ClaI. The excised gene was introduced into the expression plasmidpHD86SP13 (Dalboge et al. 1987), Biotechnol. 5, 161 ff), and introducedinto E.coli MC1061.

Extracts from bacteria containing the expression plasmid were analyzedin a IGF-1 specific RIA and by Western blot. Non of the analyzed cloneswere found to express detectable quantities of IGF-1. In E.coli smallcytoplasmic foreign polypeptides (less than 90-100 amino acids) have atendency to be breaked down as they are recognized as incorrectlysynthesized proteins. Transport of such proteins to the periplasma wherethe proteolytic activity is qualitatively different from the activity inthe cytoplasm may stabilize these polypeptides.

Example 3

Periplasmic production of AlaGlu-IGF-1.

The signal peptide used was from OmpA of E.coli. In the natural OmpAprecursor the signal peptide is processed between two alanine residues.To maintain this processing site the MetGlu-IGF-1 gene was altered by invitro mutagenesis to AlaGlu-IGF-1. This gene was introduced into anexpression vector pHD313 (Dalboge et al. 1989, Gene, 79, 325-332)containing the lambda PR promoter, and optimized in ribosome bindingsite, and the CI ts gene in such manner that the signal peptide wasfused directly to the AlaGlu-IGF-1 gene in the processing site. Theobtained plasmid pHD365 was introduced into E.coli MC1061 and culturedat 28° C. for 18 hrs. Production of IGF-1 was induced by raising thetemperature to 40° C.

Example 4

Extraction of AlaGIu-IGF-1 from E.coli.

To 4 liters of E.coli fermantation broth was added cysteine to a finalconcentration of 50 mM, and urea to a final concentration of 7M. Theculture was extracted at pH 8.0 for 30 min at 6°-10° C. The extract wascentrifuged for 30 min (4000 rpm) and the supernatant was either frozenor further processed immediately.

Example 5

Dialysis.

One liter of cell culture extract from Example 1 was diluted to 1.5liters using 0.5 liters of 7M urea and 50 mM cysteine. The dilutedextract was dialyzed by passing the extract through a hollow fibreapparatus (Nephros Presto, H.F. Organon). The flow was 100 ml per min.Change of buffer was carried out by reverse passing of 7M urea and 50 mMcysteine at pH 8.0 at a flow rate of 133 ml per min. The extract andbuffer was recycled until an equilibrium was reached. The dialysis wasterminated at ionic strength of 2.0 mS.

Example 6

Anion exchange of crude AlaGlu-IGF-1.

DE-52 (Whatmann) anion exchange material was equilibrated using 50 mMtris-HCl, 7M urea, 50 mM cystein at a conductivity of 2.0 mS. Theequilibrated anion exchange material was mixed with the crude dialyzedextract obtained in Example 2 (protein/gel) in a ratio of 1 g to 30 ml).The crude extract was absorbed for 2 hrs at room temperature or overnight at 4° C. The absorption was followed by collection of thenon-absorbed proteins by filtration on Millipore A25 filter. Thefiltrate was reabsorbed for 3 hrs at ambient temperature on FF-QSepharose, which was preequilibrated using the same buffer (protein/gelratio was 1 g/200 ml) . The quality and the quantity of the denaturedAlaGlu-IGF-1 was determined by reverse phase chromatography. The columnapplied was a Nucleosil C4, 5 μm, 4.6×250 mm column in TFA,acetonitrile. Initial condition TFA: 0.085% in HzO.sub., (buffer A) ,buffer B 0.1% TFA, 80% acetonitrile. The column was eluted using amixture of buffer A and B and a gradient from 0-5 rain isocratic 30% B,from 5 to 25 rain linear from 30-75% B, flow: 1.0 ml/min; detection:absorption at 215 nm; ambient temperature. Denatured and reducedAlaGlu-IGF-1 is seen on FIG. 2.

Example 7

Renaturation of denatured AlaGlu-IGF-I by hollow fiber dialysis

The pooled denatured and reduced batches of AlaGlu-IGF-1 obtained asdescribed in Example 3 was ajusted to a protein concentration of 0.2mg/ml using 7M urea and 50 mM cystein. The pH was adjusted to 9.5 using5M NaOH. The solution was dialyzed against a buffer consisting of 50 mMTris-Cl, 2 mM cystein, 2 mM EDTA, pH 9.5 and 25% ethanol. The dialysiswas carried out using a hollow fibre dialysis apparatus (Nephros ProstoH. F. Organon) at a flow of 100 ml/min. The dialysis buffer wasexchanged when equilibrium was obtained between solution and buffer. Theprocess was continued until a conductivity of 150 μS and a redoxpotential of 25 mV was reached. After the initial phase during whichmost of the AlaGlu-IGF-1 was folded, the solution was allowed tostabilize at 4° C. over night. 16 hrs later the reaction was terminatedby adjusting the pH to 4.0 using 5M acetic acid. After dialysis, theionic strength was 0.2 mS and the concentration of urea was estimated tobe <0.05M.

An example of the renaturation of an aliquot of denatured and reducedAlaGlu-IGF-1 is shown in FIG. 3. The peak at RT 14.86 min. is ascrambled form of AlaGlu-IGF-1 (RT 15.68) of M_(r) 7845 a.m.u. Peaksappearing later than AlaGlu-IGF-1 are probably scrambled forms of themolecule.

The redox electrode used for this step was ajusted using a 5 mMhydroquinone solution having a defined redox potential of 463 mV at pH4.0.

Reference is made to FIG. 6 concerning the hollow fiber RP-HPLC.

In the diagram of FIG. 6, the peak at RT 15.99 is AlaGlu-IGF-1. The peakat RT 15.21 is a scrambled form of AlaGlu-IGF-1 having a M_(r) of 7845a.m.u.

The renaturation of denatured AlaGlu-IGF-1 was also performed in thefollowing three manners:

1) diluting the denatured sample, 2 mg/ml, in 50 mM Tris, 2 mM EDTA, pH9.0, 25% ethanol to a final concentration of 0.2 mg/ml (BioRad assay)and letting the reaction proceed for 18 hours at 4° C.

2) desalting the denatured sample on an NAP 5 column (Pharmacia-LKB) in50 mM Tris, 1 mM Cystein, 2 mM EDTA, pH 9.0, 25% Ethanol. The elutionvolume was 1.00 ml. The concentration of protein was 0.3 mg/ml (BioRadassay) and the reaction was allowed to procede for 18 hours at 4° C.

3) dialysis of the denatured sample (Spectapor 3.5 kD cut-off) against50 mM Tris, 1 mM Cystein, 2 mM EDTA, pH 9.0, 25% ethanol at 4° C. for 18hours. The initial concentration of protein was 0.3 mg/ml.

In each experiment 200 μl of the resulting solution was acidified with30 μl of 5M acetic acid and 200 μl hereof were filtered on a Millex 0.22μm filter and subjected to RP-HPLC analysis on a Nucleosil C4, 5 μ,4.6×250 ntm column, Machery-Nagel 720059, A buffer: 0.085%trifluoroacetic acid (TFA) , B buffer: 0.1% TFA, 80% acetonitrile usinga gradient: from 0 to rain isocratic 30% B, from 5 to 25 min. linearfrom 30-75% B and a flow of 1.0 ml/min. detection at 215 nm at ambienttemperature.

Reference is made to FIGS. 7-9.

In the diagram of FIG. 7, the peak at RT 16.07 min. is AlaGlu-IGF-1. Thepeak at RT 15.1 is a scrambled form of AlaGlu-IGF-1 having a M_(r) of7845 a.m.u. Peaks appearing just later than AlaGlu-IGF-1 are probablyscrambled forms as well.

In the diagram of FIG. 8, the peak at RT 16.07 is AlaGlu-IGF-1. The peakat RT 15.10 min. is a scrambled form of M_(r) 7845 a.m.u. Peaksappearing just later than AlaGlu-IGF-1 are probably scrambled forms aswell. Peaks appearing later than 20.00 min. are probably irreversiblyscrambled or polymeric forms of AlaGlu-IGF-1.

In the diagram of FIG. 9, the peak at RT 16.21 min. is AlaGlu-IGF-1. Thepeak at RT 15.43 min. is a scrambled form of M_(r) 7845 a.m.u. Peaksappearing just later than AlaGlu-IGF-1 are probably scrambled forms aswell.

Example 8

Cation exchange of renatured AlaGlu-IGF-1.

70 ml of FF-S Sepharose (Pharmacia-LKB) were equilibrated in 25 mMNa-phosphate, 25 mM Na-citrate adjusted with 5M NaOH to pH 4.0, 25%ethanol.

2 liters of renaturation mixture obtained as described in Example 7 wereapplied to the FF-S Sepharose. The suspension was gently mixed for 30minutes at ambient temperature, and then the gel was packed in a column(3.5×20 cm²). The buffers used were

A: 25 mM Na-phosphate, 25 mM Na-citrate pH 4.0, 25% ethanol

B: 25 mM Na-phosphate, 25 mM Na-citrate pH 7.0, 25% ethanol bothadjusted using 5M NaOH.

The flow was 0.15 ml/cmz/min, detection at 280 nm, and temperature 4° C.A gradient of 0-100% B over 60 minutes was applied. The fractionscomprising AlaGlu-IGF-1 as determined by absorption at 280 nm werepooled.

Example 9

Conversion of AIaGlu-IGF-1 to IGF-1 using DAP-1.

The pool of AlaGlu-IGF-1 obtained in Example 8 was dialysed against 40mM Na-citrate, 200 mM NaCl pH 4.0 (adjusted with 5 M NaOH) at 4° C. for18 hours. The temperature was raised to 37° C. and 0.08 units ofCatepsin C (3.4.14.1. Boehringer Mannheim) were added per mg of protein.After 30 minutes the reaction mixture was applied for a RP-HPLC column.

For termination of the reaction and purification, reaction mixture wasapplied to a RP-HPLC column (Novo Nordisk A/S RP18, 15μ, 10×250 mm) in100 mM Na-phosphate buffer pH 3.0 and eluted using a step-gradient offrom 30-50% ethanol over 30-60 minutes. The flow was 0.75 ml/min,detection at 280 nm, ambient temperature.

The fractions comprising IGF as determined by absorption at 280 nm werepooled.

Example 10

Cation exchange of IGF-1.

The pooled frations of IGF-1 obtained from the RP-HPLC purification asdescribed in Example 9 was applied to a FF-S® Sepharose HR10/10 columnfrom Pharmacia-LKB. The buffers used were

A: 25 mM Na-phosphate, 25 mM Na-citrate pH 4.0, 25% ethanol

B: 25 mM Na-phosphate, 25 mM Na-citrate pH 7.0, 25% ethanol bothadjusted using 5M NaOH.

The flow was 0.15 ml/cm² /min, detection at 280 nm, temperature

A linear gradient of 0-100% B over 100 minutes was used. The fractionscomprising IGF-1 as determined by absorption at 280 nm were pooled.

Example 11

Dialysis and gelfiltration of IGF-1.

The pooled fractions of IGF-1 obtained in Example 10 were dialyzed(Spectrapor 3.5 KD cut-off) against 0.1M acetic acid at 4° C. for 18hours and then lyophilized. The lyophilized powder was dissolved in 7Murea to a concentration of 5 mg/ml and applied a Sephadex G50F column,(0.9×60 cm, Pharmacia-LKB) in 0.4M acetic acid. Flow was 0.2 ml/min,detection at 280 nm, temperature 4° C.

The fractions comprising IGF-1 as determined by absorption at 280 nmwere pooled.

The solution obtained may be lyophilized using conventionallyophilization agents to form a stable lyophilized powder. The powdermay be used for preparation of pharmaceutical preparations in a mannerknown per se for formulating pharmaceutical preparations comprisingIGF-1.

We claim:
 1. A method for the renaturation of AlaGlu-IGF-1 whereinAlaGlu-IGF-1, in a denatured and reduced form, is folded during changingthe redox potential from -40 mV to +20 to +40 mV by dialysis against abuffer containing from 20-40% v/v ethanol over a period of up to 5 hoursat a protein concentration of 0.1 to 0.6 mg/ml and a conductivity of0.15 to 0.3 mS, at ambient temperature and a pH of from 7.5 to 10.0 andfinally acidifying the mixture to a pH below
 5. 2. A method forproducing human IGF-1 comprising(i) expressing AlaLGlu-IGF-1 in amicroorganism transformed with an expression vector comprising aninducible promoter coupled directly to the DNA coding for humanAla-Glu-IGF-1 and a signal sequence providing for secretion of theexpressed AlaLGlu-IGF-1, (ii) extracting the expressed humanAlaLGlu-IGF-1 at a pH of more than 6.0 using urea in the presence of areducing agent to obtain an extract, (iii) subjecting the extract ofstep (ii) to chromatography on an anion exchange gel selected from thegroup consisting of DEAE, DE and FF-Q' in a buffer comprising urea and areducing agent adjusted to 1.8-2.2 mS and pH 7.8-8.2 to obtain anisolated human Ala-Glu-IGF-1 solution, (iv) renaturing the isolatedhuman Ala-Glu-IGF-1 of step (iii) comprising changing the redoxpotential of the isolated human Ala-Glu-IGF-1 solution from a negativeto a positive value by dialysis against a buffer containing an alcoholat a protein concentration from 0.1 to 0.6 mg/ml at ambient temperatureand a pH of from 7.5 to 10.0 and finally acidifying the mixture to a pHbelow 5 to obtain renatured human Ala-Glu-IGF-1, (v) cleaving theAla-Glu extension from the renatured human Ala-Glu-IGF-1 of step (iv)with dipeptidyl amino peptidase to obtain renatured human IGF-1, (vi)isolating the renatured human IGF-1 of step (v) using reversed-phasehigh pressure liquid chromatography and cation exchange chromatography,and (vii) gel filtrating and lyophilizing the isolated human IGF-1 ofstep (vi).
 3. A method as claimed in claim 2 wherein the microorganismis E.Coli.
 4. A method as claimed in claim 2 wherein the promoter is atemperature sensitive promoter selected from the group consisting ofλ_(Pr), λ_(PL) λ_(PR'), and the signal sequence is selected from thegroups consisting of LamB, OmpA or OmpF.
 5. A method as claimed in claim4 wherein promoter is the λ_(Pr) and the signal sequence is theOmpA-sequence.
 6. A method as claimed in claim 2 wherein the extractionof AlaGlu-IGF-1 in step ii is carried out using a buffer having aconcentration of urea greater than 4M.
 7. A method as claimed in claim 2wherein the extraction of AlaGlu-IGF-1 in step ii) is carried out usinga buffer comprising 7M urea, 50 mM Cys and pH 8.0.
 8. A method asclaimed in claim 2 wherein the chromatography of the extract from stepiii) is carried out at a conductivity of 2.0 mS at pH 8.0.
 9. A methodas claimed in claim 2 wherein the chromatography of the extract fromstep iii) is carried out as batch chromatography on DE and repeated onFF-Q®.
 10. A method as claimed in claim 2 wherein the renaturation ofAlaGlu-IGF-1 in step IV is carried out at a conductivity of 0.2 mS. 11.A method as claimed in claims 2 wherein the renaturation of step iv) iscarried out changing the redox potential linearily from about -40 mV to+25 to +30 mV over a period of 3 hours.
 12. A method as claimed in claim2 wherein the renaturation of step iv) is carried out at a conductivityof 0.2 mS, a concentration of proteins of 0.2 mg/ml, and a pH from 8.5to 9.5.
 13. The method of claim 12 in which the renaturation of step iv)is carried out at a pH from 8.5 to 9.0.
 14. A method as claimed in claim2 wherein the renaturation of step iv) is carried out at a temperatureof from 20° to 22° C.
 15. A method as claimed in claim 2 wherein therenaturation of step iv) is carried out passing the AlaGlu-IGF-1 througha hollow fiber device at a flow rate of from 25 to 250 ml/min.
 16. Themethod of claim 15 in which the flow rate is about 100 ml/min.
 17. Amethod as claimed in claim 2 wherein the acidification of step iv) iscarried out by adding 5M acetic acid.
 18. A method as claimed in claim 2wherein the cleaving of step v) is carried out using DAP-I in aconcentration of 0.08 units DAP-I per mg of protein in the presence ofNaCl, adjusting the protein concentration to approximately 1 mg/ml in a40 mM acetate buffer at pH 4.0 and at 37° C.
 19. The method of claim 2in which human AlaGlu-IGF-1 is extracted in the presence of a chelatingagent.
 20. The method of claim 2 in which the isolated humanAla-Glu-IGF-1 solution is dialyzed over a period of up to 5 hours at aconductivity of 0.15-0.3 mS and in which the alcohol is ethanol in aconcentration from 20 to 40% (v/v).